| Background and ObjectiveDiabetic retinopathy(DR) is one of the most severe complications of diabetes. And the pathogenesis of DR is very complicated. It's reported that inflammation probably play a central role in the pathogenesis of the DR. In diabetic rats, intercellular adhesion molecule-1 (ICAM-1) induced by inflammation could mediate leukocyte adhesion in the inside of retinal vascular, result in lesions of endothelial cell and blood-retinal barrier (BRB) breakdown. Sometimes, BRB breakdown is the main reason of macular edema. Concerning the researches of the relation between expression of retinal ICAM-1 and diabetic BRB breakdown, Joussen AM reversely demonstrated the expression of ICAM-1 has some relation with BRB breakdown in ICAM-1 knockout diabetic mice. In a word, chronic and low level of sub-clinical inflammation is the reason of many vascular signature lesions of DR. These researches suggested that anti-inflammation is underlying therapy for diabetic macular edema and DR.Diabetic macular edema (DME) is the certain kind of DR which can cause severe loss of vision. In the non-proliferative DR patients, DME is the main reason of reducedvision. In recently years, intravitreal triamcinolone acetonide (TA) injection has exponentially increased as a treatment option for various intraocular neovascular and edematous proliferative disorders, including DME. TA is a minimally water-soluble, artificial synthesized and long effective glucocorticoid which has strong anti-inflammation effect and a certain kind of antiangiogenic properties. TA is an effective and safe agent for intravitreal injection in conditions requiring long-term ocular steroid administration. However, the exact mechanism of TA treating DME is not clear. In vitro research showed that TA significantly decreased the paracellular permeability and down-regulated ICAM-1 expression, which suggested that reconstruction of the BRB and down-regulation of the expression of inflammatory factor should be the mechanism of therapeutic effect of TA.To investigate the effect of ICAM-1 in the BRB breakdown and supply theoretic support for the clinical use of TA in DR, this experiment ascertain the DR by observing the retinal vascular morphological changes of rat which had different diabetic duration, and studied the relation between ICAM-1 expression and BRB breakdown, and the therapeutic effect of TA for BRB breakdown in DR rat model. MethodsDR model and grouping: Healthy adult male Wistar rats were used for the study. Streptozotocin (STZ) intraperitoneal injection was used to induce DR model and 50 rats (100 eyes) with blood glucose higher than 16.7mmol/L were entered experiment. DR rats were divided into diabetes 4 months (DM4) group (25 rats) and diabetes 6 months (DM6) group (25 rats), another 25 normal rats were used as normal control (CON) group. Each group was divided into HE staining group, ICAM-1 immunohistochemcial staining group and BRB measurement group. BRB measurement group was further divided into non-TA treatment (NT) group, 1-week-TA treatment (T1) group, 2-week-TA (T2) treatment group Morphological examination: The retina was stripped after the eye balls were fixed with 4% paraformaldehyte and digested by 3%Trypsin solution for 2-3 hours, then retinal preparation was made and stained by HE method .Under microscope, observed the morphological changes and calculated the ratio of endothelial cell/pericyte.ICAM-1 expression test: The digested retinal preparation was stained by immunohistochemcial method to observe the expression of ICAM-1. The mean optic density (MOD) of endothelial cells was measured by Image-pro-plus 5.0 software to quantify the expression of ICAM-1. BRB breakdown examination: After EB was injected into the circulation for 2 hours, the rats were perfused via the left ventricle. Immediately following perfusion, both eyes were enucleated and bisected at the equator. The retinae were carefully dissected and thoroughly dried in vacuum-dry for 5 h. Evans blue was extracted by incubating each retina in formamide for 18h. The net absorbance was measured by spectrophotometry which can be calculated as the content of EB, and the content of EB was normalized for retina dry weight. The BRB change was measured by normalized EB content. Therapeutic effect evaluation: The therapy was intravitreal TA. Therapeutic effect evaluation was by BRB breakdown examination. SPSS 12.0 was used for statistical analysis. Results1. The blood glucose value of the rats: from 1 to 6 months, the blood glucose of DM4 groups were all significant higher than that of CON groups (P<0.001). However, there was no significant difference between DM4 groups and DM6 groups(P>0.05) .2. Morphological changes: ①The capillary and its caliber of CON group was regular, endothelial cell and pericyte was visible. ②The DM4 group had morphological change such as irregularity of capillary caliber, capillary occlusion, hyperplasia of endothelial cell and loss of pericyte. ③The DM6 group had more severe morphological change than that of DM4 group and acellular capillary presented. ④Semi-quantification of morphological changes (E/P): both DM4(10.37±0.82)and DM6(13.76±1.37) were significant higher than that of CON(2.94±0.39), and DM6 was higher than DM4 group (p<0.001) .3. ICAM-1 expression test: ①CON group: There was little positive expression of ICAM-1, the staining of capillary wall wasn't obvious, pericyte and endothelial cell was visible. ②DM4 group: significant positive expression of ICAM-1, the staining of capillary wall was obvious, the morphological changes was visible. ③DM6 group: thepositive expression of ICAM-1 was higher and morphological change was more severe than that of DM4 groups, and acellular capillary was visible. ?Semi-quantification of the expression of ICAM-1 (MOD): both DM4 group (103.40±7.15) and DM6 group (82.03±6.37) were significant lower than that of CON group (171.67±5.76), and DM6 was lower than that of DM4 group (P<0.001) .4. Measurement of BRB: There was no significant difference of EB content (P> 0.05) among CON groups. The EB content of the two the diabetic NT groups significantly increased (P<0.001). And The EB content of DM6 was significant higher than that of DM4 groups (P<0.01). Compared with diabetic NT group, the EB content significantly decreased in each treated diabetic groups (P<0.001), however except for the EB content of 2-week treated group which had 4 months diabetic duration had been returned almost normal (P>0.05), EB content of the rest of treated groups was still higher than CON group (P<0.05). The EB content had no significant difference among the treated groups (P>0.05).5. The correlation between the MOD of endothelial cells and the retinal EB content: There was significant correlation between the MOD of endothelial cells and the retinal EB content (r= -0.939) in NT rats.Conclusion1. DR model can be established by raising diabetic rats, which is showed as retinal vascular morphological changes and BRB breakdown, and these changes become more obvious with time prolongation.2. There is positive relation between the expression of ICAM-1 in retinal capillary and BRB breakdown.3. The inflammation plays an important role in the formation of DR at the molecule and function mechanism level.4. Intravitreal TA injection can effectively reduce the BRB breakdown, and the earlier the intravitreal TA injection use, the more effective the TA has. This result suggests that intravitreal TA injection could treat diabetic macular edema effectively.
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